Heparanase and syndecan-1: Promoters of
aggressive myeloma behavior and
targets for therapy
Ralph Sanderson, PhD
Department of Pathology
THE UNIVERSITY OF
ALABAMA AT BIRMINGHAM
Conflict of interest disclosure
Work on the heparanase inhibitor SST0001 was funded
in part by Sigma-tau Pharmaceuticals.
1
Tumor-host crosstalk regulates the microenvironment to
promote myeloma progression
Angiogenesis
Heparanase & syndecan-1
Targeting heparanase
Tumor
Gene expression
Dissemination &
Growth
Stromal components
Osteoclast
Osteolytic disease
Osteoclast
Heparanase and syndecan-1 promote
myeloma progression
Heparanase
Syndecan-1 (CD138)
Heparanase
Heparan sulfate
Core protein
Sheddase
Cell membrane
· High syndecan-1 in patients serum correlates with
high tumor mass and poor prognosis (Dhodapkar et
· High heparanase activityin patient plasma correlates with
al., 1997; Seidel et al., 2000)
high microvessel density (Kelly et al., 2003)
· Heparanase promotes myeloma growth and metastasis · Shed syndecan-1 enhances myeloma growth and
metastasis in vivo (Yang et al., 2002)
(Yang et al., 2005)
· High heparanase is an indicator of poor prognosis in
· Knockdown of syndecan-1 or heparan sulfate inhibits
growth in vivo (Khotskaya et al., 2009; Reijmers et al.,
myeloma (Mahtouk et al., 2007)
2010)
2
Heparanase and syndecan-1 regulate
the myeloma microenvironment
Angiogenesis
Clinical correlation:
Heparanase
ERK
high heparanase, high MVD
Syndecan-1
MMP-9
v3 signaling
shedding
X
VEGF
HGF
C-MET
Heparanase
HGF
HGF
HGF
X
Dissemination &
RANKL
Proliferation
X
Clinical correlation:
Osteoclast
high heparanase expression,
high HGF and high RANKL
Osteolysis
Osteoclast
SST0001: A potent heparanase inhibitor engineered
by chemically modifying heparin
Heparanase as a therapeutic target:
- there appears to be a single active heparanase in humans
- heparanase knockout mice show no obvious deficits
ST0001
Heparin
SST0001:
- potent inhibitor of heparanase activity
- non-anticoagulant
- not degraded by heparanase
3
SST0001 inhibits growth of subcutaneous
myeloma tumors
CAG tumors
Vehicle
450 ± 240 mg
P < 0.004
SST0001
X
30 ± 11 mg
18 mg/kg/day
SST0001
XX
X
X
X
0.8 ± 0.8 mg
36 mg/kg/day
RPMI-8226 tumors
Vehicle
628 ± 82 mg
SST0001
307 ± 48 mg
P < 0.001
SST0001 blocks CAG myeloma tumor growth
in human bones
PBS
SST0001
SCID-hu mouse
Human Kappa levels (tumor burden)
P< 0.003
SST0001 does not inhibit growth of
tumor cells in vitro
PBS
SST0001
4
SST0001 inhibits heparanase-enhanced syndecan-1
shedding, angiogenesis and gene expression
Saline
SST0001
MMP-9
SST0001 inhibits heparanase-enhanced syndecan-1
shedding, angiogenesis and gene expression
Saline
SST0001
VEGF
HGF
Subcutaneous tumors
MVD analysis in SCID-hu tumors
SST0001 disrupts the establishment of a microenvironment
that supports aggressive tumor growth
5
How is heparanase regulating gene expression?
Angiogenesis
Heparanase
ERK
Syndecan-1
MMP-9
v3 signaling
shedding
VEGF
HGF
C-MET
Heparanase
HGF
HGF
HGF
Dissemination &
RANKL
Proliferation
Osteoclast
Osteolysis
Osteoclast
Heparanase decreases the level of nuclear
syndecan-1 in myeloma cells
Heparanase expression :
Low
High
(CAG myeloma cells)
Immunostaining for syndecan-1
ELISA
Western Blot
Non nuclear
+ - + -
Nuclear
Nuclear
- + - +
Syndecan-1
Syndecan-1
Heparanase
Low High
6
What is the role of syndecan-1 in the nucleus?
Heparan sulfate/heparin inhibit histone acetyltransferase (HAT) activity
(Buczek-Thomas et al.)
HATs modify the N-terminal tail region of histones by acetylating key lysines
altering DNA-histone and histone-histone contacts to enhance binding of
transcriptional complexes to DNA
Abnormal HAT activity is associated with the development of cancer
Heparanase upregulates histone acetyltransferase
(HAT) activity in myeloma cells
HAT activity assay - nuclear extracts from heparanase low and high CAG cells
P <0.05
P <0.05
HPSE expression:
low high
(CAG cells)
Cell line:
U266
MM.1S
Recombinant HPSE:
-
+
-
+
Acetyl histone H3
Acetyl histone H3
Histone H3
Histone H3
7
Heparanase upregulates HAT activity
Heparanase expression (CAG cells)
Low
High
Cells in culture
Tumors in SCID mice
Immunostaining for acetylated histone H3
Syndecan-1 inhibits HAT activity in
heparanase-high cells
120
Histone H3 peptide substrate
l) 100
ity
o
80
60
activ
contr
T
40
A
of
H
(% 20
0
02468
Exogenous syndecan-1 (ug/ml)
120
Histone H4 peptide substrate
l) 100
ity
o
80
60
activ
contr
40
TA of 20
H
(%
0
02
468
Exogenous syndecan-1 (ug/ml)
8
Inhibition of HAT activity inhibits heparanase-mediated
upregulation of MMP-9 and VEGF gene expression
MMP-9 mRNA (qPCR)
VEGF mRNA (qPCR)
HPSE high
4
HPSE high
eg
an 3
HPSE low
chld 2o HPSElow
f
1
tivelaeR 0
Anacardic acid:
-
+
-
+
-
+
-
+
Inhibition of heparanase decreases HAT-regulated expression of genes
that promote aggressive tumor behavior
Angiogenesis
Heparanase
ERK
Syndecan-1
MMP-9
v3 signaling
shedding
X
VEGF
X
HGF
C-MET
HAT
HGF
HGF
X
HGF
Dissemination &
Heparanase
RANKL
Proliferation
X X
Osteoclast
Osteolysis
Osteoclast
9
Conclusions

Heparanase and syndecan-1 facilitate tumor-host crosstalk in the
microenvironment that enhances myeloma growth, dissemination, angiogenesis
and osteolysis

Heparanase modifies the tumor microenvironment by:
Enhancing shedding of syndecan-1

Shed syndecan-1 binds growth factors and facilitates signaling through
growth factor receptors

Shed syndecan-1 can activate integrins and promote their signaling
Upregulating tumor cell expression of MMP-9, VEGF, HGF & RANKL

The mechanism of regulation of gene expression by heparanase is mediated, at
least in part, by disruption of syndecan-1 localization to the nucleus resulting in
enhanced histone acetyltransferase activity

Inhibitors of heparanase represent a viable therapeutic approach for myeloma and
other cancers
Acknowledgements
Sanderson Lab
Present
Past
Li Nan
Ligong Chen
Anurag Purushothaman
Yuemeng Dai
Vishnu Ramani
Yan Huang
Joe Ritchie
Yekaterina Khotskaya
Ivonne Rivera
Veronica MacLeod
Yang Yang
Telisha Swain
Allison Theus
Funding: National Institutes of Health
Toru Uyama
Multiple Myeloma Research Foundation
Sigma-tau Pharmaceuticals, Inc.
Collaborators
Ronzoni Institute ­ Benito Casu, Giangiacomo Torri, Annamaria Naggi,
Marco Guerrini
Sigma-tau Pharmaceuticals ­ Claudio Pisano, Sergio Penco
Technion ­ Israel Vlodavsky
Weizmann Institute ­ Oded Livnah, Gali Golan
University of Arkansas for Medical Sciences ­ Bart Barlogie, Josh Epstein,
John Shaughnessy, Shmuel Yaccoby, Frank Zhan, Tom Kelly, Larry Suva
University of Wisconsin, Madison ­ Alan Rapraeger, Brian Ell
10